PROJECT SUMMARY/ABSTRACT Vascular integrity is critical for the prevention of tissue edema, thrombosis, inflammation, and hemorrhage. Identifying new factors that influence vascular integrity positively or negatively can provide important therapeutic options for maintaining vascular health. Over the last decade, my lab has taken unbiased genetic approaches in mice to generate vascular phenotypes and to discern their mechanistic causes, and we have seen a powerful theme emerge from our work: excessive proteolysis is detrimental to endothelial cells and to vascular integrity in vivo. Although proteases are well recognized for regulating sprouting angiogenesis in new vessels, there is still a gap in knowledge about their impact on the endothelium of established vessels. Therefore, the objective of this NHLBI Emerging Investigator Award is to define the contexts and the mechanisms through which proteases damage blood and lymphatic vessel integrity. We will approach this broad objective by expanding on two research areas that are supported by our published and preliminary data. First, we will examine extracellular matrix degradation in the context of protease-mediated vascular damage. We will exploit our multiple genetic models with elevated protease activity to reveal commonalities in matrix composition and fragmentation near sites of vascular rupture, and we will seek to discern the relative importance of matrix scaffolding versus matrix/endothelial cell signaling in maintaining vascular integrity in vivo. Secondly, we will focus on protease-mediated endothelial cell necroptosis. Necroptosis is a newly-identified form of cell death, and little is known about its causes or effects in the vasculature. Our preliminary data indicate that proteases can directly trigger endothelial cell necroptosis during embryonic development. We will examine the mechanism through which this occurs and will determine the contribution of protease-mediated necroptosis to various postnatal vascular diseases. The proposed research is paradigm-shifting because it considers new roles for proteases in the context of vascular integrity and because it could generate new therapeutic targets for pathologies associated with high protease activity and vascular fragility.